Malaria remains one of the leading causes of morbidity and mortality in the developing world. The disease is caused by protozoan parasites that invade and ultimately destroy circulating red blood cells of their host, leading to severe anemia and the frequently lethal syndrome of cerebral malaria. These parasites have evolved a complex mechanism of immune evasion whereby, over the course of an infection, small sub-populations of parasites arise that have an altered antigenic phenotype, thus avoiding the antibody response of the host. This process of antigenic variation and is responsible for the persistent nature of the disease as well as the waves of parasitemia frequently observed in individuals infected by P. falciparum. The variation in antigenic phenotype is the result of switches in expression between individual members of the multicopy var gene family. This family consists of approximately 40-50 genes that encode the predominant antigenic determinant, a protein called PIEMPI. Only a single var gene is expressed at a time by any given parasite, thus determining the antigenic type of the infected cell. Changes in var gene expression and the resulting antigenic variation appear to be controlled at the level of transcription. The objective of this proposal is to determine the molecular mechanisms that maintain all but a single var gene in a transcriptionally silent state. The hypothesis to be tested is that var promoters are silenced through the assembly of a condensed, heterochromatic state and that chromatin modification and remodeling regulates expression of var genes and antigenic variation in Plasmodium falciparum. The experimental design exploits the recent discovery that elements upstream of var promoters act in a cooperative fashion with a conserved intron found in all var genes to silence transcription of all but a single gene. This transcriptionally silent state can be assembled on transfected episomes containing a reporter gene flanked by a var promoter and intron. Using this episomal system, the chromatin structure and subnuclear localization of silent and active var promoters will be determined. In addition, the specific DNA elements necessary for var gene silencing and activation will be identified, and the sequence requirements for DNA elements to functions as silencers or activators elucidated.